Spectra manager

The prediction of the CD spectrum of the monomeric Pu22 G4 structure was performed using the DichroCalc [45 (link)] web server starting from the PDB file of the NMR structure deposited with PDB ID 1XAV. At first, the 1XAV.pdb file was manually edited by replacing the unrecognized “DA, DC, DG, DT” text for deoxyribonucleotides with “A, C, G, T”. Then, the edited PDB file was uploaded as the input file in DichroCalc obtaining the predicted CD spectrum file, which was edited with SpectraGryph 1.2 [68 ]. The predicted CD spectrum from the “ds” format was finally visualized in Jasco Spectra Manager (JASCO Corporation, Sendai, Japan).

The infrared spectra (wavenumber range of 4000–600 cm⁻¹) were obtained from KBr pellets containing approximately 1% (w/w) of APPL powdered sample prepared in a cylindrical piston under high pressure and vacuum. The spectrophotometer used was a JASCO model 4100. The spectra were acquired by averaging 60 interferograms at 2 cm⁻¹ resolution for each recorded spectrum. Spectral data were background corrected against a pure KBr pellet reference spectrum prior to every measurement. Initial data manipulation was performed using JASCO spectra manager. In order to prevent subjective baseline values and to enhance the resolution, the original spectra were modified by subtracting a positive multiple of its 2^nd order derivative [28 ]. The assignment of signals was done in accordance with previous studies with lignocellulosic material [29 ,30 ,31 (link),32 ,33 ].

CYP1A2 holoprotein, CPR, and cytochrome b₅ contents were spectrophotometrically measured by ultraviolet-visible spectrophotometry (Cary 300 UV-Vis spectrophotometer, Agilent Technologies, Santa Clara, CA, USA), as previously reported [27 (link),29 (link)]. Data analysis was conducted using Jasco Spectra Manager (JASCO Corporation, Sendai, Japan). Cuvettes (Sub-Micro Cells; 16.50-Q-10/Z20) were purchased from Starna Scientific, Ltd. (London, UK).

CYB content was measured spectrophotometrically according to previously reported methods with several modifications^{35 (link)}. The microsomal fraction (50 μg) was diluted to 200 μL in 100 mM potassium phosphate buffer (pH 7.4) containing 1.0 mM sodium cyanide. Ninety microliters of sample protein were added to the sample and reference cuvettes. A baseline between 400 and 500 nm was recorded using a Cary 300 UV–Vis spectrophotometer (Agilent Technologies). After the addition of 10 μL 4 mM NADH, the absorbance spectra of both cuvettes were spectrophotometrically recorded between 400 and 500 nm. All assays and measurements were performed in triplicates using a single microsomal preparation. Data analysis was conducted using a Jasco Spectra Manager (JASCO Corporation, Sendai, Japan). CYB concentrations were calculated utilising the absorbance of three separate recordings employing reduced minus oxidised difference spectra at 424 nm (185 mM⁻¹ per cm).

A JASCO NRS-3300 equipped with a CCD detector (− 69 °C) was employed for the Raman measurements. A diode laser system emitting at 785 nm wavelength, 600 lines/mm grating and an UMPLFL Olympus objective of 20× were used for recording the Raman spectra. The calibration was performed using the sharp peak of Si from 521 cm⁻¹. For the experiments, 4 mL of fruit distillates were placed in a glass vessel; the spectrum was recorded using 100 s as exposure time and 3 accumulations.
The JASCO Spectra Manager (JASCO, Easton, USA) tools were used for spectra analysis and selection of the frequency range (120–1700 cm⁻¹) before any processing of the Raman data. Then, for each sample, the average spectrum (obtained using the statistics on rows, mean process for the spectra registered in two points) was subjected to the baseline subtraction and the [0,1] normalization. These processes were realized in OriginPro 2017 (OriginLab, Northampton, USA) and allowed a fair comparison of the samples, especially of those manifesting the fluorescence phenomenon. These Raman data were further employed both for general Raman and Machine Learning studies.